The long range objective of this research is an understanding of the biochemical mechanisms that regulate the expression of human genes during differentiation. The human epidermis has been chosen as a model system, since we are able to serially cultivate these cells in vitro under conditions where many of the differentiative properties, including stratification, are retained. Other epithelial cells can be similarly cultured. The major differentiation-specific proteins of these cells are the keratins, a group of 15 closely related proteins (MW 40-70kd) that form 80 Ao cytoskeletal filaments. We've found at least two distinct multigene families encoding the keratins. Only a subset of these genes is over expressed at any one time. Commitment to terminal differentiation results in a shift in the synthesis of mRNAs encoding the small keratins (40-58kd) to those encoding the larger ones (56-67kd). We have found that epidermal cells in culture will terminally differentiate if the vitamin A in the medium is removed. In addition to causing increased stratification and s. corneum formation, removal of vitamin A reversibly regulates syntehesis of the mRNAs encoding the large keratins. Increasing the vitamin A levels leads to the expression of two keratins (52kd and 40kd) not normally seen in epidermal cells, but made by some cancerous lines and also conjunctival epithelial cells. We now propose to study the effects of vitamin A on mRNA synthesis in other epithelial cells having different programs of keratin gene expression. We will characterize patterns of keratin synthesis in these and other cell types and determine which keratins are regulated by vitamin A. Morphological and biochemical changes will be compared to assess possible functional roles for individual keratins. Clones cDNA probes for the vitamin A-regulated keratin mRNAs will be constructed, sequenced and compared to our cDNA sequences corresponding to the 46-58kd keratin mRNAs which are not regulated by the vitamin. Possible abnormal regulation by vitamin A in cancerous epidermal cells will be investigated. It is the aim of this study to elucidate the significance of the multiple keratins and the mechanism by which vitamin A differentially regulates their expression.